Method for the biochemical detection of analytes

ABSTRACT

The invention relates to a method for detecting and/or quantifying analytes from a sample on an analysis carrier that has been formatted using a digital data code. Detection fields comprising the sensor elements required for the respective detection process, together with additional data structures in a defined digital format, are provided on the analysis carrier and combined to form sequcnces of formatted structures that can be interpreted as code words. To detect and quantify an analyte in a sample, the latter is applied to the analysis carrier and the formation of signal-generating elements is initiated at locations of molecular interaction. The localisation of signal-generating elements in the respective detection fields causes a formatted structure at this location to be replaced by another. This leads to the conversion of one code word into another within the predetermined quantity of valid code words. Both code words can be sequentially read and interpreted in the predetermined format. The statement concerning a successful or unsuccessful reaction is based on a comparison of the respective code words prior to and after detection. Detection takes place using a reading device, which is preferably constructed from components of the consumer goods industry.

BRIEF DESCRIPTION OF THE INVENTION

The invention relates to a method for detecting and/or quantifyingmolecules from a sample on an analysis support formatted with a digitaldata code, wherein a detection reaction induces a change of thecodewords, and these can be sequentially read and interpreted in thepredetermined format.

Surface-based detection methods have been established for many years inthe biochemical laboratory. With the increasing demands of molecularbiological research, the need for highly parallelized and miniaturizedtechnologies for studying the binding in complex molecular mixtures isgrowing.

The known methods for detecting and quantifying target molecules from asample involve a plurality of steps, which are carried out by means ofvarious devices which are sometimes elaborate and expensive. Microarraysare one of the possible ways of analyzing a multiplicity of biologicalmolecules. Microarray technology, in which many different biologicalbiomolecules such a DNA or proteins are applied, densely packed, in apredefined pattern on a substrate surface, has now become the standardmethod for parallel analysis of biological samples. This technology isused, for example, in the analysis of gene expression, in geneticdiagnosis, in biological and pharmaceutical research and for thedetermination of genetically modified organisms in the food industry.

In microarray technology, biochemical sensor molecules such as DNA orproteins are applied to metal, glass, membrane or plastic surfaces,especially polycarbonate supports. After contact with the appliedsample, it is possible to detect the molecular interaction and usuallyto obtain information about the bound quantity and/or about the strengthof the interaction.

According to the prior art, the binding is usually detected via thegeneration and detection of an optical signal. In this case, it isconventional to use a microscope or a functionally similar device,especially a CD reader head (WO00/26677, WO00/36398). The informationabout binding which has or has not taken place at a particular site isobtained by image processing, which is typically carried out by computersoftware after analog-digital conversion in the case of microarrays witha relatively high density.

In one of the known methods, the information about binding which has orhas not taken place at a particular site is labeled by the accumulationof grains (beads) at the site of the reaction of the analyte with thecarrier-bound sensor molecule (for example EP918885 and Taton, Mirkin,Letsinger, Science 289: 1757-1760 (2000)). According to the prior art,beads bound in such a way are either identified directly as bodies orcause a chemical reaction such as a color change or a dyestuffprecipitate. These are essentially detected by photometric methods. Acamera and microscope combination is used to generate data, which can beevaluated by image analysis in the computer.

One of the disadvantages of the known methods for evaluating microarrayanalyses is the use of complicated and expensive devices and softwarefor detecting and evaluating very weak signals, for example emission bya few molecules of fluorescent dyestuffs. These readers and evaluationdevices are the result of many years of development work, use elaboratemethods such as confocal scanning microscopy, and are very expensive.These devices furthermore require special software for identifying andlocalizing molecule spots and for interpreting and integrating detectedsignals.

In the next few years, initial results from gene research will start tobe used in medical diagnosis and prognosis. Above all, simple and fastmolecular detection methods will be required for various clinicalproblems. There is therefore a need for a simple, inexpensive and atleast partly automated method for detecting and analyzing molecules incomplex mixtures. It is an object of the present invention to provide afast and simple method for analyzing and detecting analytes, whichoffers reliable interpretation of the results according to definedcriteria as well as quantitative information.

In order to achieve this object, the invention relates to a method withthe features mentioned in claim 1. Refinements of the invention are thesubject matter of dependent claims which, like the abstract, have beenworded with reference to the content of the description.

The above object is achieved according to the invention by a method inwhich a molecular interaction at a particular site on the support leadsto the generation of detectable structures, referred here as signalingelements, which can be read and interpreted in the context of the formatdefined in the form of a digital data code on the analysis support.

Detection fields with the sensor elements needed for the respectivedetection, as well as other data structures, are applied in a digitalformat on the analysis support and are combined to form sequences offormat structures which can be clearly interpreted as codewords. Inorder to detect or quantify an analyte in a sample, the latter isapplied to the analysis support and modulation of the digital signallevel on the respective detection fields is initiated with the aid of asignaling element. In this way, one codeword is replaced by anothercodeword allowed within the set of valid codewords. The informationabout a reaction which has or has not taken place is based on comparisonof the respective format structures before and after the detection. Theexemplary embodiments are a CD, a magnetic card, an optical card and abarcode card.

The method according to the invention has the advantage, over knownmethods, that elaborate two-dimensional image analysis since digitaldata are obtained. The provision of a digital information structureobviates analog signal processing, which is complicated and prone toerrors. Reliable interpretation of the results according to definedcriteria is facilitated. The analysis support and the reader, which isconstructed from standard components in the consumer goods industry, canfurthermore be adapted to one another easily as a function of theproblem, so that various tests can be developed and produced quickly andinexpensively in mass production.

Other advantages, features and possible applications of the inventionwill be described below with the aid of the detailed description andexemplary embodiments, with reference to the drawings. In the drawings:

FIG. 1: A) shows a schematic representation of an exemplary sequence offormat elements on an analysis support, which consists of blank fields5, address fields 6 (gray squares) and detection fields 7 (whitesquares), and which forms a track. The header region 8 is used forfinding and initializing the track when reading; B) before detection,the sequence of format elements represents the codeword A; C) afterdetection has taken place, the represented sequence of format elementsmay represent the word A, if no reaction takes place (I.), or thecodeword B in the event of a reaction (II.). The binary codewords A andB differ by a change of the signal level at the fourth position, whichcorresponds to a detection field.

FIG. 2: A) shows a schematic representation of the simplest embodimentof the analysis support 1, with a track 2 of format elements, sketchedlinearly by way of example, and a header region 8; B) shows a schematicrepresentation of another possible configuration of the analysis support1 with a track 2 of format elements and a header region 8, an optionalcentral hole 3 for combination of the analysis support and a CD,together with a spiral data track 4 for the data storage and software;C) shows a schematic representation of an example of another preferredembodiment of the analysis support 1 with a central hole 3, a spiraldata track 4 in the CD-R standard for the data storage and software, anda spiral track 32 of format elements in the CD-R standard; D) shows aschematic representation of another possible configuration of theanalysis support in the form of a CD 33 with a central hole 3, a spiraldata track 4 in the CD-R standard for the data storage and software, anda spiral track 32 of format elements in the CD standard.

FIG. 3: shows a schematic representation of an analysis support 1 with aplurality of tracks arranged in parallel, which consist of rows ofdetection, information and blank fields. Exemplary filling of theanalysis support 1 by means of a microfluidic plate 9 with sensorelements or analyte solutions through microchannels 10 embedded in thesupport, or otherwise spatially arranged 10, is represented.

FIG. 4: shows a schematic representation of a detection according toExample 1. A binding reaction between a sensor element 34, here anantibody, applied to the analysis support 1 and an analyte molecule 12,here a protein, from the sample is represented. The detection is carriedout in a sandwich immunoassay with the aid of a second antibody 11,which is directed against a different epitope of the protein. Acolloidal gold particle 13 coupled to the second antibody leads to thedeposition of a silver grain 14, which is used as a signaling element.

FIG. 5: shows a schematic representation of a detection according toExample 3. A binding reaction between a receptor 15 applied to thesupport 1 and a ligand 16 from the sample, which is coupled to a hapten17, here biotin, is represented. After addition of a mixture ofstreptavidin 18 and biotinylated ferretin 19, signaling elements areproduced in the form of molecular complexes 20.

FIG. 6: shows signaling elements for the example of polystyrene beadswith a diameter of 1 μm, which are detected according to two differentmethods and interpreted in binary form. A) shows an image recorded by aCD reader head; B) shows the same beads recorded by fluorescencemicroscopy; C) shows a three-dimensional representation of the data fromA), which can be interpreted in binary form.

FIG. 7: A) shows a simplified schematic representation of an opticalsystem for detecting the reflection signal, which represents aconventional CD reader head, consisting of a laser (L) 25, a detectorfor focal adjustment and signal detection (D/F) 27, a focusinginstrument 28 and a semisilvered plate 29. The analysis support 1 withthe silvering 30 is represented in the beam path in the side view; B)shows a simplified schematic representation of an optical system fordetecting the transmission signal, consisting of a CD pickup fromExample A), the actual detector for the transmission measurement (D) 26and optionally an additional focusing instrument 31. The analysissupport 1 with the silvering 30 is represented in the beam path betweenthe focusing instruments 28 and 31 in the side view.

FIG. 8: shows a schematic representation of an analysis support, onwhich the detection fields are applied as parallel strips so that theycan be read using a barcode reader. A) The individual detection fields23 are arranged in relatively large detection regions the form of teststrips 22. They may be fitted in a test unit together with addressfields 21 which, for example, contain information about testspecifications, codings (dongle) or product identification. After adetection reaction with analytes from different samples 1 and 2,different patterns of signaling elements are obtained on the teststrips, and these can be read using a barcode reader; B) The test strips22 may consist of a plurality of detection fields 24, which are arrangedorthogonally to the main reading direction and, for example, may containgraded concentrations.

FIG. 8: shows an image of fine lines in the micrometer range, which areattributable to an antibody-antigen reaction with subsequent silverdeposition. A) shows an analog image recorded by a CD reader head; B)shows analog image lines from A), read along the horizontally dashedline; C) shows digital processing of the image line from B), producedfrom the analog signal by using a threshold criterion, identified by thehorizontal line in B).

METHOD FOR BIOCHEMICAL DETECTION OF ANALYTES DETAILED DESCRIPTION OF THEINVENTION

The present invention comprises the following essential components,which in combination constitute the preferred use of the methodaccording to the invention:

-   -   an analysis support, consisting of a base support and blank,        information and detection fields applied thereon, which are        formatted in a digital data code;    -   sensor elements, applied to a defined pattern of the detection        fields;    -   a standard protocol for carrying out specific interactions        between sensor elements and analytes from the sample, and for        the formation of signaling elements on the detection fields;    -   a reader for the detection of signaling elements on the        detection field in the context of the predetermined digital        format, together with control means and evaluation software.

The components essential for carrying out the method according to theinvention will be explained in detail below:

Coding

The analysis support is formatted with a defined digital data code. Theformat is dictated by the specific technical embodiment and thedetection system which is used, or in general the reader. For example,the format is established by the respective characteristics of pits andlands in CD technology, “high” and “low” levels in digital electronics,dots and dashes in Morse code etc. In the case of the detection systemsknown from the CD technology, for example, a defined geometry, lengthand arrangement of pits and lands on an optical disk, according to thePhilips Red Book standard, determine the respective format.

The coding describes the control mechanism according to which theinformation is processed. A code is the sum of all valid codewords, eachcodeword being defined by a unique sequence of the predetermined formatstructures. Coding generally contains additional rules such as redundanterror-correction information, for example cross summation orinterleaving, as specified for example by the Red Book standard in theCD industry.

The most common formats in the consumer goods industry use a binarycode. This case will be described in detail below as a preferredembodiment. A binary code is given by a particular sequence of “0” and“1” levels (or “high” and “low” levels) with a defined length. In thecase of the present invention, the binary code consists of sequences offormat structures, which are defined by “blank-field” and“information-field” format elements (FIG. 1A) with particular signallevels and signal lengths. Information fields may be address fields ordetection fields.

In a particularly preferred embodiment, the sequences of format elementson the analysis support form one or more tracks. In a preferredembodiment, a track is divided into four different subregions (FIG. 1A):

1. Header region. Here, the track is found and initialized (tracking).

2. Blank fields. The breaks are essential in order to determine theposition of the detection field to be read, by counting or viaaddressing. The blank fields have an invariant level (“0” or “1”) and avariable length.

3. Address fields. These contain structure information and positioninformation, with which it is possible to assign the signal obtained bythe molecular interaction to a defined substance. The address fieldshave an invariant level (“1” or “0”) and can have different lengths. Theaddress fields and the blank fields have inverse levels. If the blankfields have a “0” level, for example, then the level of the addressfields is “1”, or vice versa.

4. Detection fields. The sensor elements are applied here. The detectionfields are characterized by a variable level (“0” or “1”) and can havedifferent lengths. A level change from “0” to “1” or from “1” to “0” ona detection field indicates that detection of the analyte has takenplace (see below).

Any conceivable defined arrangements of format elements in one or twodimensions are possible, and are therefore covered by the invention.

It is a fundamental concept of the present invention that the signalingelements on the detection fields can be adapted to the predeterminedformat, and that the result of respective biochemical detection can beobtained from comparison between the respective sequences of the formatstructures before and after the analysis is carried out.

According to the invention, the vocabulary of the code consists of alimited number of codewords with a predetermined length, which are madeup of the “blank-field” and “information-field” format elements withdefined signal levels and signal lengths. Each word may contain one ormore detection fields. Undefined words are not allowed and areidentified as errors by the interpreter software. Advantageously, acertain number of codewords have no detection fields and are used, forexample, for separating and/or addressing sizable code blocks. Thenumber of tests applied to a support can be scaled in a simple way bycombination or concatenating the sequences of format elements.

In order to determine the detection result, a defined sequence of formatelements defines two allowed codewords A and B, which differ by a changeof the level from “0” to “1” or from “1” to “0” on at least onedetection field (FIG. 1B). Before detection, the levels on the detectionfields are such that the sequence of format elements represents thecodeword A, for example. A detection reaction causes replacement of “0”by “1”, or vice versa. The codeword A is therefore converted into thecodeword B. If no reaction has taken place on the detection fields, therespective sequence of format elements still represents the codeword A(FIG. 1C). The result is interpreted by comparison between therespective sequences of format structures before and after the analysis.

Detection evaluation on an analysis support according to the inventionwill be explained using the example of Morse code. Suppose that Morsecode is implemented on the analysis support by a predetermined sequenceof “low” and “high” levels. Assume that the “low” level always has thelength 1 and is represented by the symbol “0”. In this example, it isused only as a spacer mark. The “high” level selectively has the length1 or 3, however, and is consequently represented by “1” or “111”. Assumethat the codeword A is defined by the sequence “1 0 1 1 1 0 1” and thecodeword B is defined by the sequence “1 0 1 0 1 0 1” (FIG. 1B). Bothwords have the same total length of 7, but the level at the fourthposition, which is here intended to correspond to a detection field onthe analysis support, is different. A binding reaction on the detectionfield at the fourth position converts the “1” level at this point to a“0” level. The allowed word A is therefore changed into the allowed wordB (FIG. 1C). The detection result is derived by simple comparisonbetween the output word A obtained before the detection and the word Bafter the detection. The word A will be read if no reaction takes place(“negative test”), and the word B will be read in the event that areaction has taken place (“positive test”). A different codeword C, oran undefined word, would be picked up in the scope of the interpretationand identified as an error. This feature of the method canadvantageously be used for quality control.

This indirect obtaining of sequential binary data, based on coding, hassubstantial advantages over the known methods. First, elaboratetwo-dimensional image analysis is avoided. Secondly, the selection of adigital code establishes a digital information structure, which obviatescomplicated and error-prone analog signal processing. Thirdly, thecoding of binary signals allows error detection and correction.Fourthly, standard modules or complete devices from the consumer goodsindustry may be used for the detection and evaluation.

A format which is conventional in the consumer goods industry ispreferably used on the analysis support according to the invention. Thismay, for example, be audio CD, optical disk, CD-R, CD-RW or MO (ECMA154, ISO/IEC 10090), CD-ROM (ECMA 130, ISO/IEC 10149), DVD-R (ECMA 268,ISO/IEC 16449) or subsequent standards.

The invention is not restricted to formats which use a binary code.Other formats based on digital code systems are also conceivable, andare therefore covered by the invention.

Multidimensional codes, such as two-dimensional barcodes, are a directextension and are therefore included in the subject matter of thepresent invention. It may be advantageous to use such coding forbiochemical detections because it allows parallel data processing, errorcorrection and obtaining of redundant information. Parallel dataacquisition, for example by means of a camera or CCD chip, is advantagein this case because the absolute positions can thereby be determinedand interrogated directly. This contrasts with serial reading, in whichposition determination needs to be carried out with the aid of marks,addresses or synchronization.

Analysis Support

The analysis support of may have a polygonal, round, oval or other two-or three-dimensional shape. In a particularly preferred embodiment ofthe invention, a substrate which is similar in geometry and handling toconventional magnetic cards is used as a support (FIG. 2A). Magnetic andchip cards have enjoyed widespread use owing to their practical size andease of handling. The essential features are their robustness and thepossibility of accommodating a limited amount of information on a smallspace. In one common variant, the magnetic cards contain a linearlyarranged data strip at a constant distance from one of its outer edges.In combination with the simple reader, this leads to simple mechanicalhandling—the card can even be swiped by hand through a reader. Becausemagnetic cards can be produced inexpensively in large numbers and thereaders are easy to manufacture, this embodiment of the invention makesit possible to construct readers which are orders of magnitude lessexpensive in production and simpler to use than known equipment for thedetection of biological analytes.

Magnetic storage media, optical cards, a barcode card (FIG. 8) orcombinations thereof may also be used as analysis supports. According tothe invention, the external shape is not restricted to the usual cardstandards. A conventional CD (FIG. 2D) or derivatives thereof, forexample CD-R, DVD, may likewise be used as analysis supports. Existingformatting of format structures on the analysis support mayadvantageously be utilized, which obviates involved and expensivereprogramming.

The detection area may be part of the support or accommodated on aseparate auxiliary support. Software, databases, signatures and otherinformation, besides the sensor elements, for example testspecifications, protocols for carrying out the respective test, etc. maybe fitted in any desired configuration on the same support. Any desiredcombinations of conventional data storages, such as magnetic strips,card chips, barcodes, CD (FIGS. 2B, 2C and 2D), CD-ROM, audio CD or CD-Rmay be integrated in the support.

If the sequences of format elements on the analysis support form one ormore tracks, and the detection, information and blank fields areequivalently dimensioned, and each have an area of 5×5 μm², then alength of 35 μm for the test unit is obtained in the Morse code exampledescribed above. When a plurality of such test units are arrangedsuccessively, they form a track (FIG. 1A). Assuming a track length of 6cm, an individual analysis number of up to 2000 is achieved in onetrack. It is therefore possible either to carry out up to 2000 differenttests on one analysis support or, by using graded concentrations, toobtain extensive statistics about a small number of detections.

It is possible to have a plurality of tracks of detection, informationand blank fields arranged in parallel per support (FIG. 3). Thisarrangement offers the advantage of increasing the individual testnumber and parallelizing the test procedure and readout of the results.Furthermore, for example, the filling of individual detection fieldswith sensor elements and/or a sample can be parallelized via amicrofluidic plate.

Linear, curved, radial, spiral and circular, or other geometrical oreven stochastic arrangements of detection, information and blank fieldsare also possible, of course, and are therefore covered by theinvention.

Polymer plastics with various physicochemical properties adapted to theanalytical task, as well as glass, semiconductors, metals, metal alloys,ceramics, hybrid materials or combinations of these substances may beused as the support material. A particularly advantageous embodiment incombination with optical reading methods employs supports made of glass,transparent plastics or polymer materials, and especiallyoptical-quality polycarbonate as used in the CD and DVD industry.

In order to produce an analysis support, basic formatting is firstapplied to the base support. For each specific test, the sensor elementsnecessary for the respective detection are then applied in a predefinedpattern on the format elements provided as detection fields.

Sensor Elements

Any substances which may be of use for biochemical or medical detectioncan be used as sensor elements. Examples include sugars, steroids,hormones, lipids, proteins, in particular monoclonal or polyclonal orrecombinant antibodies, peptides, antigens of any type, haptens, DNA,RNA, as well as natural and artificial derivatives thereof, inparticular aptamers and PNA, but also organic-chemical active agentlibraries as used, for example, in pharmacological research anddevelopment. Cells, microorganisms, viruses or parts thereof,preparations and extracts from biological materials, metabolites and thelike may equally well be used as sensor elements. Further chemical,biological, organic or inorganic elements with sensor characteristicsmay equally well be employed, and are therefore covered by theinvention.

According to the invention, a multiplicity of different sensor elementsmay advantageously be applied to a substrate surface. The sensorelements of one type are in this case respectively applied to defineddetection fields in a spatially limited way. The extent of the detectionfields in one or two dimensions may be less than 10 μm, preferably lessthan 2 μm and particularly preferably less than 1 μm. Besides the sensorelements, it is also possible for other molecules or signaling elements,which are used for calibrating or standardizing the analyses, to beapplied according to a predefined arrangement on neighboring detectionfields.

Solutions by which sensor elements can be bound to the support surfacewithout detrimentally affecting their functionality are known to theperson skilled in the art from the prior art. The sensor elements may becovalently or noncovalently bound to the support surface or applied tothe support surface. The sensor elements may be applied mechanically tothe support surface, in particular by droplet application, for examplewith the aid of inkjet printing or spotting, or by means of lithographicmethods according to the prior art. The wetting of the detection fieldswith the sensor elements may also be carried out by means of channels,preferably microchannels or microfluidic networks (FIG. 3). Simpleimmersion of the analysis support in a liquid bath containing the sensorelements is also possible, after selective activation of detectionfields or passivation of blank fields on a surface-activated support. Aspin-coding method may likewise be used, for example in order toextensive activation of the surface.

In another advantageous variant, material which is transparent at aparticular wavelength is used for the analysis support. Thelight-guiding properties of the support may be used according to theinvention in order to couple or synthesize particular molecules atpredefined sites via position-selective guiding of light. Synthesiscontrolled by electric fields directly on the support is likewiseconceivable.

The sensor elements may, for example, be covalently linked by bindingamino, thio or phospho groups, which are already present or have beenspecially introduced, to a terminal-group-functionalized silanizedsupport surface. Alternatively, biotinylated sensor elements may bespecifically immobilized on the support surface by means of streptavidincoating.

Signaling Elements

It is a fundamental concept of the present invention that the physicalparameters of the signaling elements, such as size, shape and signallevel, can be adapted to the format structure applied in the form ofblank fields and address fields. This contrasts with the usualprocedure, in which a format structure and device are developed so as tomatch the given signal. The constraints such as positioning anddimensioning of the format elements and of the signaling elements aredictated by the coding.

Any resonant processes (absorption, fluorescence, phosphorescence,plasmon resonance, quenching etc.) and nonresonant processes(reflection, diffraction, scattering etc.) from spectroscopy may be usedfor the signaling. Alternatively, electromagnetic effects (piezo,resonance shift, capacitance change, Hall effect, magnetic effects,electrical charge displacement etc.) may be used for the signaling.Chemical processes such as silver deposition, precipitation of oxides,oxidation or reduction of reagents, electroplating and the like may alsobe employed for the detection. Other chemical, physical or biologicalsignalers may equally well be used, and are therefore covered by theinvention.

A variety of commercially available substances and bodies, which canconstitute or form detectable structures, may be used as signalingelements. They may be advantageously be microspheres (beads) (FIG. 6) ofany shape and size, such as metal, magneto, silica orfluorescence-labeled beads, fluorescent or radioactive labels as well asmolecular complexes or aggregates, layers of precipitates or dyestuffs.

In a preferred alternative embodiment of the optical detection, silvergrains are formed on an initiator, in particular an electron donor suchas metal-molecule or metal grains, coupled to analyte molecules, at thesite of the interaction, which is usually binding (FIG. 4). In anotherpreferred variant of the optical detection, molecular complexes areformed in a reaction between two or more different binding partners, forexample one avidin or streptavidin, and another multiply biotinylatedsubstance (FIG. 5). Biological objects of suitable size such as cells,bacteria, pollens, virus particles or parts thereof may advantageouslybe used as signaling elements.

In another advantageous embodiment, the resulting detectable structuremay also be produced by initiation of a chemical reaction with anothersubstance. To this end, another substance is applied to defined pointson the support, in addition to the sensor element which is intended tointeract with the sample to be analyzed. This substance is convertedinto a detectable structure at the site of the reaction by theinteraction between an analyte and the associated sensor element. Forexample, an enzyme such as horseradish peroxidase (HRP) coupled to theanalyte may initiate an enzymatic conversion of the substrate localizedon the support into a signaling element through the binding of theanalyte to the corresponding sensor element. The reaction is facilitatedeither by spatial proximity between the enzyme and the substrate, or byrelease of the substrate into the solution, during the interactionbetween the sensor element and the analyte or directly thereafter.

The properties after an interaction between a sensor element and ananalyte give rise to detectable structures in the form of signalingelements, and the signal levels resulting therefrom correspond to thepredetermined digital format on the support. These structures may bedimensioned by various methods known to the person skilled in the art.For example, saturation in the formation of signaling elements may beachieved through the stoichiometric ratios of the substanceconcentrations which are used, so that no further effective growth ofthe signaling element takes place after an intended size is reached.Alternatively, the reactions leading to the formation of detectablestructures may be blocked in a time-controlled way. In particular,blocking substances such as inhibitors for enzymatic reactions,competitors such as biotin in the example of FIG. 5, or substances whichbreak down free reactants, for example specific proteases, may be usedfor this. In a particularly preferred embodiment, the detectionreactions take place in a continuous flow system, so that the reactantsinvolved can be flushed away from the reaction sites with a buffer afteran experimentally determined optimum reaction time, which is necessaryin order to form detectable structures with intended dimensions. Ifcatalytic reactions are involved, they may also be stopped by removingor blocking the catalyst. In the case of photodependent reactions,switching off the light source also leads to controlled termination ofthe reaction.

Detection fields and signaling elements with dimensions smaller than 10μm, preferably smaller than 2 μm, and particularly preferably smallerthan 1 μm, are advantageously formed. Any lower limit on theminiaturization is due only to the resolution of the detection unit.

Establishment of the reaction conditions and selection of the reactantsleads to detectable structures in an interaction between a sensorelement and an analyte, which can be read and digitally interpreted inthe data format previously applied to the support. In this way, theresult can be interpreted directly as “positive test” or “negative test”by comparing the signals before and after the detection with one anotherin the context of the predetermined formatting.

A molecular interaction may lead either to the generation of anadditional signal or to reduction of the signal level due to thesignaling element. The appropriate contrast methods, i.e. change of thesignal level due to the signaling element, should in this case beselected as a function of the standard protocol for the respectivebiochemical test, the measurement method and the analysis support.

Successful detection of the analyte is characterized by a modifiedsignal structure, i.e. a signal level change from “low” to “high”, orfrom “high” to “low”, and unsuccessful detection of the analyte ischaracterized by an unmodified signal. With the aid of the followingexamples, the four possible contrast methods for the preferred variantof the optical detection by means of reflection and transmissionmeasurements will be described by way of example.

1. “Low”-“high” transition in a reflection measurement. This situationis encountered, for example, in the case of a weakly reflecting analysissupport surface and a reflective signaling element. Such contrast may,for example, be produced by means of a black surface (“low” level) andby silver precipitation induced by the positive test as a signalingelement (“high” level). A similar principle is used in black-and-whitephotography.

2. “High”-“low” transition in a reflection measurement. Here, forexample, binding or production of a scattering body (“low”) as asignaling element on a silvered support surface (“high”) may beinitiated by a successful reaction. Such a scattering body may, forexample, be a bead or a silver grain.

3. “Low”-“high” transition in a transmission measurement. This situationis encountered, for example, in the case of a silvered analysis supportand a signaling element in the form of a window. The window (“high”) maybe produced by local etching, induced by a molecular interaction withthe analyte, and associated removal of the mirror layer (“low”) from thedetection fields. This method is comparable with the typicallithographic etching methods in semiconductor physics.

4. “High”-“low” transition in a transmission measurement. This situationis encountered, for example, in the case of an unsilvered andtransparent analysis support and a reflective or scattering signalingelement. Such a contrast may be produced, for example, with the aid ofan uncoated glass plate (“high”) by means of silver precipitation as asignaling element (“low”), similarly to black-and-white photography. Thelight is scattered by the silver grains and the transmission is therebyattenuated.

A light-scattering bead represents another possibility for a signalingelement.

Quantitative information can be obtained according to the invention bymultiple determinations and/or by graded concentrations of the sensorelement and/or analyte and subsequent statistics.

Reader

The invention furthermore relates to a reader which is optimized for therespective analysis support, and which, in an advantageous embodiment,allows semiautomatic or automatic positioning, passage and reading ofthe support. The support may also be automatically scanned.

An existing product from consumer electronics is preferably used as thereader, or new devices from common detection and mechanical units incombination. CD, DVD, magnetic-card or barcode readers may be mentionedhere as examples.

In a preferred embodiment, the reader corresponds essentially to amanual magnetic-card reader in terms of its mechanics and handling. Inthis case, either the reader is installed mechanically fixed and theanalysis support is passed through it, or the analysis support is placedfixed and the reader is moved linearly over the support by usingcorresponding mechanics.

In an advantageous embodiment of the invention, the reader is based onoptical detection. The reader consists essentially of a “photoelectricbarrier” with one or two sides. Corresponding to the signal type, asuitable reader head is used for the detection. The optical reader may,for example but not exclusively, have the embodiments described below.

A particularly preferred embodiment employs a commercially available CDreader head, which operates by reflection (FIG. 7A). Alternatively, amodified CD reader head is used which, in contrast to the reader unitused in CD players, can operate with transmitted light as well, or onlywith transmitted light (FIG. 7B). In this case, the silvering necessaryin a conventional CD may be obviated, which leads to inexpensiveproduction of the analysis support.

The analysis support may advantageously be oriented in both directions,i.e. with the front side, i.e. the side coated with sensor elements,toward or away from the reader unit, for example a CD reader head. Theappropriate orientation depends on the biochemical detection protocol tobe carried out, the respective signaling element, general constraints,for example coverage of the test areas or the base material of theanalysis support, and the detection method. In the case of a reflectionmeasurement, for example, it is advantageous for a silvered front sidewith a light-scattering signaling element to be oriented toward theoptical reader unit, because a scattering element on a rear side wouldnot be picked up in this case. For encapsulated liquid delivery by meansof channel structures on the analysis support, in the case of reflectivesignaling elements, however, orientation of the front side away from thereader unit is advantageous. Otherwise, the light would need to passthrough the channel structures for a reflection measurement, so thatcontamination and interference effects due to the solutions in thestructures could occur.

Another preferred embodiment employs a conventional barcode reader, whenthe detection fields are arranged on the support as parallel strips in apattern similar to a barcode (FIG. 8A). In a refinement of thisembodiment, a multiplicity of parallel detection fields with identicalsubstances may be fitted next to one another on such a test strip. Inthis case, the test results are read as a function of angle, for exampleorthogonally to the reading direction of the barcode reader (FIG. 8B).This gives rise to the possibility of multiple measurement in adetection being carried out, in order to obtain statistical information.Alternatively, a test strip subdivided in such a way may have detectionfields to contain different, gradually arranged concentrations of sensorelements, so that quantitative information is possible. In this way,detections with micro and macro dimensions can be carried out in anydesired combination on a support.

The parallel tracks of detection fields on an analysis support may alsoadvantageously be read in parallel with multi-focus optics, for example7 parallel light beams, as already used sometimes in modern CD-ROMdevices. The individual photodiodes of a CD reader head, which are usedfor the tracking, may also be employed as a detector. An HF filter willbe used as an isolator in this case, in order to separate the readsignal from the tracking signal. In principle, the detection may also becarried out two-dimensionally, for example with a camera. This isparticularly advantageous in the case of an extended arrangement offormat elements, for example in the case of a 2D barcode.

Just as suitable as CD reader heads for detectors are their successors,as used in CD-ROM, CD-R and DVD readers, as well as magneto-optical andmagnetic detectors, linear CCD arrays or photodiode arrays.

The invention is not restricted to optical data acquisition. Inparticular, magnetic detection methods may be used as analysis supportsbecause of their widespread use in consumer electronics. Magnetic cardsin banking as well as tape drives and hard disks in the computerindustry may be mentioned here as examples. The associated readersystems can be used as reader devices after minor adaptations to thegeometry of the respective analysis support.

Inside the reader, a reader head uses a senso-electrical transducer togenerate analog signals, which can be processed directly by digitalconversion and subsequent microprocessor logic for use in an evaluationsystem.

The analysis support is evaluated in four steps: a) measurement of thesignal level, for example by means of a CD pick up; b) digitization ofthe analog signals, i.e. production of a digital signal sequence withthe aid of a threshold criterion; c) interpretation of the digitalsignal stream with the aid of predefined format structures; d)comparison with the signal sequences allowed within the formatstructure.

The digitization of the analog signal level is carried out in a standarddevice, for example in an electronic module with standard A/D converterlogic. Image processing is obviated, because binary threshold criteriaare provided by the device (FIG. 9). Synchronization of the dataacquisition is carried out automatically with the aid of thepredetermined sequence of detection, address and blank fields. In a morerefined embodiment, special features of the signaling elements may beemphasized or suppressed with the aid of digital signal processing (DSP,microprocessor etc.), in order to obtain clear result information. Thismay be done, for example, by means of filtering or frequency analysis,by taking into account only signals with specific characteristics of thesignaling elements and the format structures during the evaluation.

The incoming datastream is checked in an interpreter as to whether thechronological sequence of signal levels is permitted within thepredetermined data format. Direct checking and error detection isthereby implemented. The signal sequence to be analyzed also containsthe position information needed for the test. The received signalsequence is interpreted by comparison with the allowed sequences. Asequence which is allowed within the coding but is unexpected clearlyindicates an error.

A series of individual tests on an analysis support can be carried outusing a plurality of sequences of format elements arranged in rows. Inthe case of a CD, the format elements together with the detection fieldsare introduced directly into the track of pits and lands on the CD. Thesignal levels, the signal lengths, as well as the interpretation of thesignal sequences, are given here by the Philips “Red Book” standard. Theevaluation steps a) to c) are implemented in any standard CD player. Thetest is then evaluated directly by comparison of the predetermined inputcodeword with the output codeword which is read.

For evaluating the tests accommodated on the support, the subsequentcomputer system requires special software which is tailored to therespective analysis support. In an advantageous embodiment, thissoftware may be accommodated in the data regions present on the samesupport. If the analysis support is correspondingly designed in terms ofshape and function, it is possible to use readers such as floppy-disk orremovable hard drives, CD-ROM players or comparable devices and theirsuccessors, with which the support is in principle compatible, in orderto read the software. Advantageously, a plurality of readers may also bepresent in one device. The analysis software may also access databasesoptionally present on the support, in order to gain access to standardvalues which are required in the context of the specific analyses.

Applications

Owing to the ease of handling and the rapid and precise informationabout the detection results, the present invention can be usedparticularly advantageously in biochemical or biomedical detectionmethods. The invention furthermore relates to the substance combinations(“kits”) required for a detection. The following may be mentioned indetail:

-   -   hospital laboratories and genetic counseling for routine        parallel diagnosis of genetic predispositions;    -   specialist medical practices for sensitive detection of        pathogens, bacteria, viruses, autoimmune and tumor diseases,        immunological overreactions and metabolic diseases;    -   pharmaceutical industry for mass screening to find        pharmaceutically relevant active agents and quality control in        active-agent production;    -   molecular biology in fundamental research for characterizing        interactions in complex molecular mixtures;    -   plant genetics and hybrid development to cultivate commercially        useful plants for agriculture, parallel analysis of plant        features for example gene data;    -   environmental analysis for determining soil quality factors,        contamination, occurrence and levels of microorganisms;    -   veterinary practice for simultaneously carrying out biochemical        tests and unequivocal identification of the animal on site with        the aid of an identification mark, for example a implantable        microchip which can be read without contact through the skin,        and which can be read with a simple transportable device;    -   point-of-care use for carrying out diagnostic methods on site,        i.e. for a specialist practitioner or for outpatients, e.g. for        routinely performed tests such as measuring blood sugar, blood        lipid (LDL/HDL), determining immuno status etc.    -   food industry for detecting genetically modified organisms,        monitoring microbiological or biochemical processes and quality        control;    -   agriculture for developing agrochemicals.

Other uses of the analysis platform according to the invention arelikewise possible, and are therefore included in the subject matter ofthe invention.

EXEMPLARY EMBODIMENTS EXAMPLE 1 Detection of C-reactive Protein by Meansof Silver Precipitate Formation

A polycarbonate support is cleaned in water/ethanol (1:2) usingultrasound. A monoclonal antibody (Clone 5 (4C28), HyTest, Finland)against human C-reactive protein (CRP) is then printed onto definedregions of the support by means of a polydimethylsiloxane (PDMS) padusing a microcontact printing method. The pad and the support are inthis case arranged with respect to one another, with the aid of analigning unit, so as to ensure accurate positioning to within 5 μm. Thesupport is then blocked for 30 min with a solution of 1% bovine serumalbumin (BSA). After washing with buffer (PBS, 10 mM Na phosphate, 145mM NaCl, 4 mM KCl, pH 7.4), the support is incubated for 30 min in atest sample containing the CRT protein. After rewashing with PBS, thesubstrate is incubated for 30 min with a second, biotinylated monoclonalantibody directed against a different epitope of CRP (Clone 7 (4C29),HyTest, Finland; in 1% BSA in PBS, 1:300 of the stock solution). Inorder to detect the binding which has taken place, by means of asandwich immunoassay (FIG. 4), the support is incubated for 20 min withan antibiotin antibody conjugated to 1 nm colloidal gold particles,which is diluted by 1:400 in 1% BSA in PBS. A silver solution is addedafter washing. In an advantageous embodiment, this consists of 110 mgsilver lactate, 850 mg hydroquinone (alternative: pyrogallol), 2.55 gcitric acid monohydrate, 2.35 g trisodium citrate made up to 100 ml withwater, which is freshly prepared immediately before the reaction. Inorder to ensure more uniform precipitate formation, the silver solutionmay be provided with further additives, for example UV blockers andreaction inhibitors such as gum arabic. After 2-4 min in the dark, thereaction is stopped by washing with distilled water and subsequentlydeveloped for 5 min with 3% (w/v) sodium thiosulfate in water.Alternatively, silver acetate may be used instead of silver lactate; inthis case, the reaction does not take place in the dark, but for 15 minunder normal daylight. In another advantageous embodiment, a commercial“silver enhancement” solution (e.g. from Sigma-Chemie, Munich) known tothe person skilled in the art from immunohistology may be used instead.After rewashing with distilled water, the support is read in anessentially commercially available CD reader head.

EXAMPLE 2 Detection of C-reactive Protein by Means of Alkali Phosphatase

A support is prepared and processed in a similar way to Example 1.Instead of the antibiotin antibody, however, a streptavidin-alkaliphosphatase conjugate is now added (Sigma-Chemie, Munich, 20 min, in 1%BSA in PBS, 0.5 mg/ml) and then washed. For signal development, thesubstrate is incubated for 10 min with ELF-97 phosphatase substrate(Molecular Probes, 5 mM in AP buffer: 150 mM NaCl, 1 mM MgCl_(2,) 1%BSA, 100 mm Tris-HCl, pH 9.5) and then thoroughly washed. The substrateconverted by the enzyme precipitates at the site of the reaction.Alternatively, other substrates and other detection enzyme complexes maybe used, for example alkali phosphatase with BCIP-NBT from Sigma-Chemie;streptavidin-peroxidase conjugate from Roche with 4-chloro-1-naphtholfrom Sigma-Chemie; peroxidase with DAP/Co from Sigma-Chemie.

EXAMPLE 3 Detection of Proteins via the Formation of Molecular Complexes

Biotinylated antibodies against a protein of interest are mixed with apatient's serum sample, which was previously diluted by 1:3 in PBS (seeabove). The mixture is centrifuged for 10 min at 13000×g, and thesupernatant is applied to a detection support which, in a proceduresimilar to that in Example 1, is coated in particular places with anantibody that binds a different epitope on the protein to be detectedfrom the serum. After thirty minutes of incubation at room temperatureand washing three times in PBS, a freshly prepared mixture of corestreptavidin and biotinylated ferritin in PBS cooled to 4° C. is appliedto the detection support. The ferritin was in this case biotinylatedwith a kit according to the prior art, so that on average 4-10 biotinmolecules are bound per ferritin tetramer. Within an incubation time oftypically 30 minutes, when the level of the protein of interest has acertain value, large crosslinked complexes are formed at the placeswhere the support is coated with the corresponding antibody (FIG. 5).These complexes can be detected optically, and their arrangement issubsequently read and detected in a similar way to that in Example 1.

EXAMPLE 4 Detection DNA Sequences via the Formation of SilverPrecipitates

The DNA oligonucleotides, which have an amino group on the end, areimmobilized on aminated polycarbonate supports(aminopropyltriethoxysilane, Fluka) by means of standard methods, forexample Crosslinker BS3, Pierce. The single-stranded DNA is used as asensor molecule for the binding of target-sequence DNA, which isbiotinylated in the PCR reaction. After hybridization, the target DNAcan be made visible by means of streptavidin-colloidal gold a and silverprecipitation reaction, and detected with the aid of a CD reader head.

1. A method for detecting and/or quantifying at least one analyte in asample on an analysis support, at least one defined sequence of fieldsbeing applied to the surface of the analysis support, characterized inthat a subset of the fields constitute detection fields; a signal of onetype is present on each field; signals of at least one defined sequenceof fields can be interpreted as a digital codeword; sensor elements areapplied to the detection fields in a controlled way; analytes arebrought in contact with the analysis support for the purpose ofmolecular interaction with the sensor elements on the detection fields;signaling elements are localized on the detection fields when amolecular interaction has taken place; one type of signal on thedetection field where the molecular interaction has taken place isreplaced by another type of signal in a predetermined way by thelocalization of signaling elements; after the replacement of a signal ofone type by a signal of another type on at least one detection fieldwithin a defined sequence of fields, the interpretation of this sequenceof fields gives a different codeword than before the molecularinteraction; comparison of the codeword read after the detection withthe known codeword before the detection gives the detection result. 2.The method as claimed in claim 1, characterized in that any replacementof a signal of one type by a signal of another type on a non-detectionfield within a defined sequence of fields is identified as an errorduring the interpretation.
 3. The method as claimed in one of thepreceding claims, characterized in that the signals on the fields can beread and interpreted in a format known from digital storage technology.4. The method as claimed. in one of the preceding claims, characterizedin that the signaling elements are designed and localized so that theycan be read and interpreted in a format known from digital storagetechnology.
 5. The method as claimed in one of the preceding claims,characterized in that a certain number of codewords have no detectionfields and are used for separating and/or addressing sizable codeblocks.
 6. The method as claimed in one of the preceding claims,characterized in that the sensor elements of one type can beunequivocally assigned to at least one defined detection field on theanalysis support, and vice versa.
 7. The method as claimed in one of thepreceding claims, characterized in that the fields on the analysissupport a shaped as spots, strips, circles or spirals, or have anothergeometrical shape.
 8. The method as claimed in one of the precedingclaims, characterized in that individual fields on the analysis supportare arranged in the form of a spot matrix or a circular, spiral,strip-shaped, linear or other geometrical or stochastic structure. 9.The method as claimed in one of the preceding claims, characterized inthat sequences of defined fields which represent codewords are arrangedsuccessively in the form of a track on the analysis support.
 10. Themethod as claimed in one of the preceding claims, characterized in thatthe tracks are arranged circularly, spirally, linearly or in anotherdefined way on the analysis support.
 11. The method as claimed in one ofthe preceding claims, characterized in that biologically activesubstances such as sugars, steroids, hormones, lipids, proteins, inparticular monoclonal or polyclonal or recombinant antibodies, peptides,antigens of any type, haptens, DNA, RNA, as well as natural andartificial derivatives thereof, in particular aptamers and PNA,organic-chemical active agent libraries, cells, microorganisms, virusesor parts thereof, preparations and extracts from biological materials,metabolites and the like can be used as the sensor elements.
 12. Themethod as claimed in one of the preceding claims, characterized in thatany resonant processes such as absorption, fluorescence,phosphorescence, plasmon resonance, quenching etc., and nonresonantprocesses such as reflection, diffraction, scattering etc., fromspectroscopy can be used to generate the signals.
 13. The method asclaimed in one of the preceding claims, characterized in thatelectromagnetic effects such as piezo, resonance shift, capacitancechange, Hall effect, magnetic effects, electrical charge displacementetc. can be used to generate the signals.
 14. The method as claimed inone of the preceding claims, characterized in that microspheres of anyshape and size, such as metal, magneto, silica or fluorescence-labeledbeads, fluorescent or radioactive labels as well as molecular complexesor aggregates, layers of precipitates, or dyestuffs can be used assignaling elements.
 15. The method as claimed in one of the precedingclaims, characterized in that biological objects such as cells,bacteria, pollens, virus particles or parts thereof can be used assignaling elements.
 16. The method as claimed in one of the precedingclaims, characterized in that bodies and coatings, in particular metalgrains, are formed as signaling elements on an initiator, in particularan electron donor, coupled to analyte molecules, at the site of theinteraction.
 17. The method as claimed in one of the preceding claims,characterized in that a binding, polymerization, precipitation,deposition or color reaction, or other chemical or biological reactions,are used to form signaling elements.
 18. The method as claimed in one ofthe preceding claims, characterized in that the signals generated by thesignaling elements are digitized by means of a threshold criterion. 19.The method as claimed in one of the preceding claims, characterized inthat the dimensions of the signaling elements can be adapted to thedimensions of the detection fields.
 20. The method as claimed in one ofthe preceding claims, characterized in that the signaling elements aredesigned so that one and only one signaling element of is localized oneach detection field.
 21. The method as claimed in one of the precedingclaims, characterized in that the fields and the signaling elements canhave dimensions smaller than 10 μm, preferably smaller than 2 μm and inparticular smaller than 1 μm.
 22. The method as claimed in one of thepreceding claims, characterized in that the analyte in the sample isquantified with the aid of calibration fields, defined thresholdcriteria and/or statistics via multiple determination.
 23. The method asclaimed in one of the preceding claims, characterized in that asynchronization track with standardized substances, surface coatings orother structures for calibrating the reader and the detection is appliedto the analysis support.
 24. The method as claimed in one of thepreceding claims, characterized in that standard substances for positiveor negative controls are applied to defined detection fields and/or toadjacently or successively arranged rows of such detection fields. 25.The method as claimed in one of the preceding claims, characterized inthat different concentrations are sensor elements of one type areapplied to defined detection fields and/or to adjacently or successivelyarranged rows of such detection fields.
 26. The method as claimed in oneof the preceding claims, characterized in that any desired combinationof conventional data stores, such as magnetic strips, card chips,barcodes, CD-ROM or CD-R, are integrated on the analysis support. 27.The method as claimed in one of the preceding claims, characterized inthat the software, databases, signatures of other information with anydesired configuration is present on the analysis support.
 28. The methodas claimed in one of the preceding claims, characterized in thatencoding or identification of the detection to be carried out on theanalysis support is present on the analysis support, in the same formator in another separately applied format.
 29. The method as claimed inone of the preceding claims, characterized in that the fields aredesigned and arranged so that they can be read by means of acommercially available barcode reader.
 30. The method as claimed in oneof the preceding claims, characterized in that the analysis support iscomparable in its physical features, such a shape, material, opticaldensity and material thickness, as well as handling, to a magnetic cardknown from mass storage technologies.
 31. The method as claimed in oneof the preceding claims, characterized in that the analysis support iscomparable in its physical features, such a shape, material, opticaldensity and material thickness, as well as handling, to a CD, CD-ROM orDVD known from mass storage technologies, or successors thereof.
 32. Themethod as claimed in one of the preceding claims, characterized in thatthe fields are arranged in the form of a spirally applied CD data trackon the analysis support.
 33. The method as claimed in one of thepreceding claims, characterized in that one or more writable data tracksare applied to the analysis support.
 34. An analysis support,characterized in that it has at least one of the features described inthe preceding claims.
 35. A device for reading the analysis supportdescribed in claim 34, characterized by the following features:instruments for transmitted-light and/or incident-light detection;instruments for magnetic or electrical detection; an instrument forautomatically finding the detection fields; an instrument for manually,semiautomatically and automatically feeding the analysis support throughthe device, together with suitable mechanical guidance; an instrumentfor recording analog signals; an instrument for digitizing analogsignals; an instrument for time- and position-resolved synchronizationof the recording of analog and/or digital signals; an instrument forerror correction when reading and/or interpreting signals.
 36. A kit,containing the essential substances for production of the analysissupport described in claim
 34. 37. A kit, containing the essentialsubstances for carrying out one or more detections on an analysissupport described in claim 34.